US Environmental Protection Agency
Office of Pesticide Programs

Office of Pesticide Programs

Microbiology Laboratory

Environmental Science Center, Ft. Meade, MD

Standard Operating Procedure for
Germicidal and Detergent Sanitizing Action of
Disinfectants Test

SOP Number: MB-27-00

Date Revised: 01-16-13


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SOP No. MB-27-00
Date Revised 01-16-13
Page 1 of 19

SOP Number

MB-27-00

Title

Germicidal and Detergent Sanitizing Action of Disinfectants Test

Scope

This SOP describes the methodology used to determine the efficacy of
food contact sanitizers against two organisms, Staphylococcus aureus
and Escherichia coli. The methodology is based on AO AC method
960.09 (Germicidal and Detergent Sanitizing Action of Disinfectants -
see reference 15.1), revision date 01/09/13 (pending publication by
AOAC International).

Application

For product evaluations under the Antimicrobial Testing Program
(ATP), a study protocol is developed which identifies the specific test
conditions for a product sample such as contact time, dilutions,
neutralizers, etc.





Approval Date

SOP Developer:



Print Name:

SOP Reviewer



Print Name:

Quality Assurance Unit



Print Name:

Branch Chief



Print Name:





Data SOP issued:



Controlled copy number:



Date SOP withdrawn:




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SOP No. MB-27-00
Date Revised 01-16-13
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TABLE OF CONTENTS
Contents	Page Number

1.

DEFINITIONS

3

2.

HEALTH AND SAFETY

3

3.

PERSONNEL QUALIFICATIONS AND TRAINING

3

4.

INSTRUMENT CALIBRATION

3

5.

SAMPLE HANDLING AND STORAGE

3

6.

QUALITY CONTROL

3

7.

INTERFERENCES

3

8. NON-CONFORMING DATA

3

9.

DATA MANAGEMENT

3

10.

CAUTIONS

3

11.

SPECIAL APPARATUS AND MATERIALS

4

12.

PROCEDURE AND ANALYSIS

7

13.

DATA ANALYSIS/CALCULATIONS

12

14.

FORMS AND DATA SHEETS

12

15.

REFERENCES

13


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1. Definitions

Abbreviations/definitions are provided in the text.

2. Health and
Safety

Follow procedures specified in SOP MB-01, Laboratory Biosafety. The Study
Director and/or lead analyst should consult the Material Safety Data Sheet for
specific hazards associated with products.

3. Personnel
Qualifications
and Training

Refer to SOP ADM-04, OPP Microbiology Laboratory Training.

4. Instrument
Calibration

Refer to SOP EQ-01, EQ-02, EQ-03, EQ-04 and EQ-05 for details on method
and frequency of calibration.

5. Sample

Handling and
Storage

Refer to SOP MB-22, Disinfectant Sample Preparation, and SOP COC-01,
Chain of Custody Procedures.

6. Quality Control

For quality control purposes, the required information is documented on the
appropriate form(s) (see section 14). It is critical to maintain the highest
standards of good laboratory practices and aseptic technique during all
manipulations and handling of stock cultures.

7. Interferences

1.	Contamination of stock and test cultures will negatively impact
disinfectant efficacy testing.

2.	Any level of contamination in treated or numbers control plates that
interfere with reading the results will invalidate the test.

3.	Prior to use visually inspect all agar plates and discard any plates with
evidence of contamination.

4.	For contamination following the incubation phase, if contamination is
evident and interferes with the enumeration of the test organism, record as
a contaminant(s). Data from only one dilution may be used to calculate
the final CFU/plate.

8. Non-
conforming
Data

1.	Sterility and/or viability controls do not yield expected results.

2.	Management of non-conforming data will be specified in the study
protocol; procedures will be consistent with SOP ADM-07, Non-
Conformance Reports.

3.	For a valid test numbers control counts must fall between 7.0 - 8.0 logs.

9. Data

Management

Data will be archived consistent with SOP ADM-03, Records and Archives.

10. Cautions

1.	Verify the volume of dilution blanks, and neutralizer tubes in advance and
adjust accordingly.

2.	Media indicated in section 11.2.a and 11,2.b for rehydrating lyophilized
cultures are specified in the ATCC Product Information Sheet. Upon
purchase of new organisms, verify that media requirements have not


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changed by checking the new ATCC Product Information Sheet.

3.	Strict adherence to the protocol is necessary for the validity of the test
results. Microbiological methods are technique-sensitive and technique-
oriented; thus, exact adherence to the method, good laboratory practices,
and quality control are required for proficiency and validity of the results

4.	Do not allow the pipette to touch the neck or side of the flask during the
addition of the culture suspension.

5.	Plating should be completed within 1 hour after the initiation of serial
dilutions.

6.	For spread plating: ensure that the entire surface of the agar plate is dry
before adding inoculum. If necessary, leave the agar plates uncovered in
the biological safety cabinet (BSC) until the moisture has been completely
absorbed into the medium.

7.	Diluted product must be used within three hours of preparation unless
otherwise specified.

8.	Detergents used in washing glassware may leave residues which are
bacteriostatic. Test for inhibitory residues on glassware periodically
according to SOP QC-03, Glass Washing and Detergent Residues Test.

9.	Sterility and performance of medium can be verified if the subculture
medium is an adequate growth medium as stated in SOP MB-10, Media
and Reagents Used in Microbiological Assays Including Performance
Assessment and Sterility Verification.

11. Special

Apparatus and
Materials

1.	Test organisms. Escherichia coli (ATCC No. 11229) and Staphylococcus
aureus (ATCC No. 6538) obtained directly from a reputable supplier
(e.g., ATCC).

2.	Culture media (e.g., nutrient agar). Note: Commercial dehydrated media
made to conform to the recipes provided in AOAC Method 960.09 may be
substituted.

a.	Trypticase Soy Agar (TSA). Prepare according to manufacturer's
instructions. Used for the generation of frozen stock cultures for S. aureus.

b.	Tryptic Soy Broth (TSB). Prepare according to manufacturer's instructions.
Used for the generation of frozen stock cultures for S. aureus.

c.	Nutrient broth. Boil 5 g beef extract (paste or powder), 5 g NaCl,
and 10 g peptone (anatone) in 1 L H2O for 20 minutes and dilute to
volume with de-ionized water; adjust to pH 6.8  0.1. Filter through
paper (Whatman No. 4, or equivalent), place 10 mL portions in 20 x
150 mm test tubes, and steam sterilize 20 min at 121C. Use this
broth for the preparation of Nutrient agar plates.

d.	Nutrient agar (AOAC): Dissolve Bacto agar to 1.5% in nutrient broth
and adjust to pH 1.2-1 A. Steam sterilize for 20 min at 121C.


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Dispense into plates. Media is used for the generation of frozen stock
cultures for E. coli.

e.	Nutrient agar -A (NA-A): Boil 3 g beef extract, 5 g peptone and 15 g
salt free agar in 1 L de-ionized water. Dot not use premixed
dehydrated media. Dispense 10 mL portions in 20 x 150 mm tubes or
20 mL portions in 25 x 150 mm tubes and steam sterilize for 20 min at
121C. Slant tubes after sterilization and let cool. Media is used for
daily transfers of test cultures.

f.	Nutrient agar - B (NA-B): Boil 3 g beef extract, 5 g peptone and 30 g
salt free agar in 1 L de-ionized water. Dot not use premixed
dehydrated media. Steam sterilize for 20 min at 121 C. Temper
media prior to dispensing 20-30 mL portions into sterile Petri dishes.
Media is used for development of the final test culture.

3.	Subculture media: choose the appropriate recovery agar and neutralizer to
inactivate the test chemical, for example:

a.	Trypone glucose extract agar plus Neutralizer (TGEA-N): Combine
24 g of dehydrated media with 975 mL de-ionized water and 25 mL
stock neutralizer if necessary. Steam sterilize for 15 min at 121C.
Media used for the recovery of test organisms from treated samples.

b.	Tryptone glucose extract agar (TGEA): Prepare according to the
manufacturer's instructions. Plating medium is used for enumeration
of numbers control samples.

4.	Cryoprotectant solution (TSB with 15% glycerol). Suspend 7.5 g tryptic
soy broth in 212.5 mL de-ionized water. Add 37.5 g glycerol and stir
until dissolved; boil to dissolve completely. Dispense into bottles and
autoclave for 15 minutes at 121C. Solution is used for the preservation
of frozen stock cultures.

5.	Neutralizer Stock Solution (NSS): Mix 40 g Lecithin, 280 mL polysorbate
80 and 1.25 mL 0.25 M phosphate buffer stock solution. Dilute with de-
ionized water to 1 L and adjust pH to 7.2. Dispense in 100 mL portions
and steam sterilize for 20 min at 121C.

6.	Neutralizer blanks (NB): For use with < 200ppm quaternary ammonium
compounds. Mix 100 mL neutralizer stock solution, 25 mL 0.25 M
phosphate buffer stock solution, and 1675 mL of de-ionized water.
Dispense into appropriate size vessel and sterilize for 20 min at 121C.
Alternate neutralizers may be used as necessary.

7.	Phosphate buffer stock solution (PBSS): 0.25M. Dissolve 34 g KH2PO4
in 500 mL de-ionized water in 1 L volumetric flask. Adjust pH to 7.2 with


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1 N NaOH, and dilute to 1 L volume mark. Sterilize by filtration.

8.	Phosphate buffer solution (PBS). Add 1.25 mL of phosphate buffer stock
solution and 8.75 g of NaCl to a volumetric flask; fill with de-ionized
water to the 1000 mL mark and mix. A pH of approximately 7.0 is
desirable. Sterilize by filtration or autoclaving at 121C for 15-20 min.
Alternative PBS formulations with the same pH may be used (e.g., dilute
commercially prepared 10x PBS solution to lx using de-ionized water).

9.	Phosphate buffer dilution water stock solution (PBDW-SS). Dissolve 34.0
g of potassium dihydrogen phosphate (KH2PO4) in 500 mL de-ionized
water. Adjust pH to 7.2  0.2 with 0.1 N NaOH or 0.1 N HC1 and bring to
1000 mL with de-ionized water. Alternative phosphate buffers with the
same pH may be used (e.g., commercially prepared 10X PBS solution).

10.	Phosphate buffer dilution water (PBDW): Add 1.25 mL of 0.25 M
phosphate buffer stock solution to 1 L de-ionized water. Dispense into
appropriate size vessel and sterilize for 20 min at 121C. Used for
numbers control and dilution blanks.

11.	Tween-80 (polysorbate 80).

12.	PBS (lx) with 0.1% Tween 80 (PBS + T80): Add 100 mL PBS 10x
solution and 1 mL Tween 80 to a volumetric flask; fill with de-ionized
water to the 1000 mL mark and mix thoroughly. Sterilize by filtration.

13.	Sterile water. Use reagent-grade water free of substances that interfere
with analytical methods. Any method of preparation of reagent-grade
water is acceptable provided that the requisite quality can be met.

Reverse osmosis, distillation, and deionization in various combinations all
can produce reagent-grade water when used in the proper arrangement.
See Standard Methods for the Examination of Water and Wastewater and
SOP QC-01, Quality Assurance of Purified Water for details on reagent-
grade water.

14.	Blood Agar (BAP). Commercially purchased plates. Media used for the
presumptive identification of the test microbes.

15.	Mannitol Salt Agar (MSA). Combine 111 g of dehydrated media with 1 L
de-ionized water and mix thoroughly. Steam sterilize for 15 min at
121C. Media used for the presumptive identification of the test
microbes.

16.	Xylose lysine deoxycholate agar (XLD). Commercially purchased plates.
Media used for the presumptive identification of the test microbes.

17.	Glassware. For sanitizer efficacy and numbers control, use 250 mL wide
mouth Erlenmeyer flasks. For measuring sanitizers and dilution blanks


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use 100 mL graduated cylinders. For glassware used to prepare test
chemical, refer to SOP MB-22.

18.	Petri dishes. Sterile plates, 20 mm x 100 mm in size.

19.	Recirculating chiller unit. For maintaining specified temperature of the
test chemical (capable of maintaining 25  1C).

20.	Spectrophotometer. Calibrated.

21.	Test tube racks. Any convenient style.

22.	Transfer loops. Make 4 mm ID single loop at end of 50-75 mm (2-3 in.)
Pt or Pt alloy wire No. 23 B&S gage or 4 mm loop fused on 75 mm (3 in.)
shaft (available from Johnson Matthey, West Chester, PA 19380, USA).
Fit other end in suitable holder. Bend loop at 30 angle with stem.
Commercially available 4 mm ID transfer loops may also be used.
Volumetric transfer devices may be used instead of transfer loops (e.g.,
micro volume pipette).

23.	Timer. For managing timed activities, any certified timer that can display
time in seconds.

24.	Micropipettes. For performing culture transfers and serial dilutions.

25.	Whatman No. 2 filter paper. Sterile.

26.	Gram stain kit.

27.	Indole reagent. For the identification of E. coli.

28.	Catalase Reagent. For the identification of S. aureus.

29.	Staphaurex Reagent Kit. For the identification of S. aureus.

12. Procedure and
Analysis

Prior to testing, perform the neutralization assay to determine if the prescribed
neutralizer is appropriate for the test chemical.

12.1 Test Culture
Preparation

Refer to Attachment 2. A for preparation of the frozen stock cultures. Refer to
MB-02: Tracking of Test Microorganisms, Section 12 for the tracking and
transfer notations for the test microbes. Reinitiate new frozen stock cultures
every 18 months with a new lyophilized culture.

a. Defrost a single cryovial of frozen stock culture at room temperature
and briefly vortex to mix. Streak one loopful of the thawed frozen
stock onto a NA - A slant and incubate at 36  1C for 24  2 h.
Only one daily transfer is required prior to the initiation of the final


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test culture.

b.	For the final test culture, add 5 mL of PBDW to a NA - A slant
(daily culture). Using a sterile loop, dislodge growth from agar
surface. Collect mixture and transfer to a flask containing 99 mL of
PBDW. Mix throughly. Add 200 |iL of the mixture to inoculate a
minimum of 5 NA - B plates and to create a bacterial lawn.

Incubate at 36  1C for 24  2 h.

c.	Record all culture transfers on the Organism Culture Tracking Form
(see section 14).

12.2 Test Culture
Harvesting

a.	After incubation, add a minimum of 5 mL of PBS + Tween 80 to
each plate. Using a sterile rod, gently dislodge culture from agar
surface, avoid disrupting agar. Combine culture from all plates and
mix thoroughly.

b.	Filter culture through sterile Whatman No. 2 filter paper using a
vacuum source; collect filtered culture into a sterile vessel.

c.	Standardize the test culture, if necessary using PBDW to achieve a
final test culture microbe population of 1.0 x 109 CFU/ mL and 1.0 x
1010 CFU/mL (9-10 logio/mL).

12.3 Test chemical
Sample
Preparation

a.	Prepare test chemical sample per SOP MB-22.

1)	Ready-to-use products are tested as received; no dilution is
required.

2)	If hard water is required as the diluent, prepare synthetic hard
water as described in SOP MB-22.

b.	Equilibrate water bath and allow it to come to 25  1C or the
temperature specified (1C). Prepare the test chemical dilutions
within 3 h of performing the assay.

c.	Dispense 99 mL aliquots of the diluted test chemical or ready-to-use
product into sterile wide mouth Erlenmeyer flasks. Prepare one
flask per test microbe, for each germicide to be tested.

d.	Place flasks in the equilibrated water bath for approximately 10 min
to allow test solution to come to specified temperature.

e.	Record the temperature of the water bath and recirculating chiller
before and after testing on the Germicidal and Detergent Sanitizing
Action of Disinfectants Method Test Information and Culture
Preparation Sheet (see section 14).

f.	In addition, prepare a similar flask containing 99 mL of PBDW to
use for numbers control for each organism.


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12.4 Test Procedure

a.	Add 1 mL of test culture to the test flask as follows:

b.	Whirl flask, stopping just before suspension is added, creating
enough residual motion of liquid to prevent pooling of suspension at
the point of contact with test sample.

c.	Add suspension midway between center and the inner edge of the
flask with tip of pipette slightly immersed in test solution. Avoid
touching the neck or side of flask during addition. Swirl flask to
thoroughly mix contents. NOTE: The flasks containing test culture
x test chemical are referred to as the treated samples.

d.	At 30  3 seconds after addition of the test culture, transfer a 1 mL
aliquot from the test flask (test culture x test chemical) to a tube
containing 9 mL neutralizer blank and mix well. This corresponds to
10"1 dilution tube. Record timed events on the Germicidal and
detergent Sanitizing Action of Disinfectants Method Time
Recording Sheet for Transfers (see section 14).

e.	Treated samples plating. From 10"1 tube (i.e., 9 mL neutralizer
tube inoculated with 1 mL of exposed culture)., plate four 1 mL
aliquots and four 0.1 mL aliquots onto TGEA - N plates, for a total

1 2

of 8 plates per treated sample. This will result in 10" and 10"
dilutions respectively. Incubate plates at 361C for 24-30 hours.

f.	Following incubation, count colonies on TGEA-N plates. Counts
over 300 are recorded as TNTC. Record plate counts on the
Germicidal and Detergent Sanitizing Action of Disinfectants
Method Results Sheet (see section 14).

12.5 Numbers
Control

a.	Numbers control assay should be conducted within 5 minutes of
completion of treated samples.

b.	In a sterile 250 mL wide mouth Erlenmeyer flask containing 99 mL
sterile PBDW, add 1 mL of the test culture (same test culture used
for the treated samples) as follows:

c.	Whirl flask, stopping just before suspension is added, creating
enough residual motion of liquid to prevent pooling of suspension at
the point of contact with test sample.

d.	Add suspension midway between center and the inner edge of the
flask with tip of pipette slightly immersed in test solution. Avoid
touching to the neck or side of flask during addition. Swirl flask to
thoroughly mix contents.

e.	Numbers control plating. Within 30 seconds of addition of test
culture, transfer 1 mL aliquot from the test flask (test culture x
PBDW) into a tube containing 9 mL of neutralizer and mix well.
This corresponds to 10"1 dilution tube. Make serial 10-fold dilutions
in 9 mL PBDW, out to 10"6.


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f.	Plate four 1 mL aliquots and four 0.1 mL aliquots from the 10"6
dilution tube onto TGEA plates, for a total of 8 plates per numbers
control sample. This will result in 10"6 and 10"7 dilutions
respectively.

g.	Incubate plates at 361C for 24-30 hours.

h.	Following incubation, count colonies on TGEA plates. Counts over
300 are recorded as TNTC. Record plate counts on the Germicidal
and Detergent Sanitizing Action of Disinfectants Method Results
Sheet (see section 14).

i.	For a valid test, the numbers control must fall between 7.0 - 8.0
logio/mL.

12.6 Sterility
controls

a.	Neutralizer blank - plate 1 mL from a previously unopened tube
onto a TGEA plate.

b.	Sanitizer - Plate 1 mL of the product onto a TGEA plate.

c.	Diluent - Plate 1 mL of the diluent onto a TGEA plate.

d.	PBDW - Plate 1 mL of the diluent onto a TGEA plate.

e.	Incubate all plates at 361C for 24 -30 hours, record results.

f.	To be considered valid, no growth should be observed on any of the
sterility controls.

12.7 Neutralization
Confirmation
Test

a.	A neutralization confirmation test should be performed prior to or
concurrently with the sanitizer evaluation assay. The neutralization
assay must demonstrate the recovery of a low level test organism
population (e.g., 10-100 CFU/mL) in the recovery media (i.e.
TGEA-N and TGEA).

b.	Test culture Titer (TCT) . Add 0.1 mL of the test organism, which
has been serially diluted to target between 10-100 CFU/mL to 10
mL of PBDW and mix thoroughly. Dilutions 10"4 and 10"5 should
provide the range of 10-100 CFU/mL. Hold the mixture for a
minimum of two minutes. Plate 0.1 mL aliquots in duplicate onto
TGEA. Incubate plates at 361C for 24-30 hours and record
number of colonies.

c.	Neutralization Confirmation Treatment (NCT). Add 1 mL of the
test chemical to 9 mL of the prescribed neutralizer and mix
thoroughly. Within 30 seconds, inoculate the sample with 0.1 mL
of the test organism used for the TCT. Mix thoroughly. Hold the
mixture for a minimum of two minutes. Plate 0.1 mL aliquots in
duplicate onto TGEA-N. Incubate plates at 361C for 24-30 hours
and record number of colonies.

d.	Neutralization Toxicity Treatment (NTT) . Add 0.1 mL of the test


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Page 11 of 19



organism used for the TCT to 10 mL of the prescribed neutralizer
and mix thoroughly. Hold the mixture for a minimum of two
minutes. Plate 0.1 mL aliquots in duplicate onto TGEA-N. Incubate
plates at 361C for 24-30 hours and record number of colonies.

e.	Plates that have colony counts over 300 are reported as TNTC.
Record the counts on the Neutralization Confirmation Assay Results
Sheet (see section 14).

f.	Neutralization Results and Calculations: In order to demonstrate
effective neutralization of the sanitizer, differences between
treatments should not exceed 1.0 log (e.g., TCT minus NCT).

To calculate CFU/mL for Neutralization Confirmation Test use the following
equation.

CFU/mL = (avg. CFU for 10~x ) + (avg. CFU for 10~y )

10 x and 10 v

where 10 x and 10 v are the dilutions plated. Two plates per dilution are
plated for all treatments. Alternatively if only one dilution is plated use the
same equation for only one dilution. Use counts of 0 to 300 for calculation
purposes. Score counts >300 as TNTC (too numerous to count).

NOTE: A spreadsheet will be used for data analysis and calculations
associated with the neutralization confirmation assay.

12.8 Results

For the test chemical to be considered efficacious, a > 5 log reduction is
required with a contact time of 30 seconds. Retestingguidance: For tests
where the product meets the performance standard and the numbers control
mean logio density value is above 8.0, no retesting is necessary. For tests
where the product fails to meet the performance standard and the numbers
control mean lotio density is below 7.0, no retesting is necessary.

12.9 Confirmatory
Steps for Test
Microbes

a.	Presumptive identification of the test microbes will be conducted
when results are indicative of a failing efficacy evaluation or when
results are inconclusive or have discrepancies.

b.	Representative growth from one plate per treatment sample should
be confirmed by Gram stain and growth characteristics on general
and selective media.

c.	Gram stains are performed on smears taken from the treatment
sample plates. For the additional confirmatory tests, a smear of
culture from each selected treatment sample plate is streaked onto
BAP and selective media appropriate for the test organism and
incubated for 18-24 hours at 36  1C. See Attachment 1 for Gram


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stain reactions, cell morphology, and colony characteristics on solid
media.

d.	If characteristics on general and/or selective media are unusual, then
further confirmation will be conducted by using the following: rapid
indole test for E. coli and Catalase and Staphaurex test for S. aureus.
Refer to the appropriate Confirmation Flow Chart for S. aureus, and
E. coli (see Attachment 3).

e.	If confirmatory testing determines that the identity of the organism
was not the test organism the entry on the results sheet must be
annotated to indicate a contaminant was present.

13. Data Analysis/
Calculations

Calculations will be computed using a Microsoft Excel spreadsheet (see
section 14). Both electronic and hard copies of the spreadsheet will be
retained.

To calculate CFU/mL, use the following equation.

CFU/mL = (avg. CFU for 10~x ) + (avg. CFU for 10~y )

10 x and 10 v

where 10 x and 10 v are the dilutions plated. Four plates per dilution are plated
for treated samples and numbers control samples. Use counts of 0 to 300 for
calculation purposes. Score counts >300 as TNTC (too numerous to count).

a.	Calculate the mean logio density (LD) for number control plates

b.	Calculate the mean logio density (LD) for treated samples plates.

c.	Calculate the logio reduction (LR) for treated samples:

Logio reduction = mean logio numbers control - mean logio treated sample

14. Forms and Data
Sheets

1.	Attachment 1: Typical Growth Characteristics of strains of S. aureus and
E. coli

2.	Attachment 2: Culture Initiation Flow Chart for S. aureus and E. coli

3.	Attachment 3: Confirmation Flow Charts for S. aureus and E. coli

4.	Test Sheets. Test sheets are stored separately from the SOP under the
following file names:

Organism Culture Tracking Form MB-27-00 Fl.docx

Test Microbe Confirmation Sheet (Quality MB-27-00 F2.docx
Control)

Germicidal and Detergent Sanitizing Action of MB-027-00 F3.docx
Disinfectants Method: Test Information and
Culture Preparation Sheet


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Germicidal and Detergent Sanitizing Action of MB-27-00 F4.docx
Disinfectants Method: Serial Dilution/Plating
Tracking Form

Germicidal and Detergent Sanitizing Action of MB-27-00 F5.docx

Disinfectants Method: Titer of Final Test culture

Form

Germicidal and Detergent Sanitizing Action of MB-27-00 F6.docx
Disinfectants Method: Results Sheet

Germicidal and Detergent Sanitizing Action of MB-027-00 F7.docx

Disinfectants Method: Time Recording Sheet for

Transfers

Germicidal and Detergent Sanitizing Action of MB-27-00 F8.docx
Disinfectants Method: Test Microbe
Confirmation Sheet

Germicidal and Detergent Sanitizing Action of MB-27-00 F9.docx
Disinfectants Method: Neutralization
Confirmation Assay - Neutralization
Confirmation Control

Germicidal and Detergent Sanitizing Action of MB-27-00 FlO.docx

Disinfectants Method: Neutralization Toxicity

Control

Germicidal and Detergent Sanitizing Action of MB-27-00 F1 l.docx
Disinfectants Method: Neutralization
Confirmation Assay - Test culture Control

Germicidal and Detergent Sanitizing Action of MB-27-00 F12.docx
Disinfectants Method: Neutralization
Confirmation Assay Results Sheet

15. References

1.	Official Methods of Analysis. 2013. 18th Ed., AOAC
INTERNATIONAL, Gaithersburg, MD. Method 960.09: Germicidal and
Detergent Sanitizing Action of Disinfectants. Revised First Action 2013.
Pending publication by AOAC International.

2.	Package Insert - Gram Stain Kit and Reagents. Becton, Dickinson and
Company. Part no. 882020191JAA. Revision 07/2011.

3.	Package Insert - Catalase Reagent Droppers. Becton, Dickinson and
Company. Part no. L001237. Revision 06/2010.

4.	Package Insert - Staphaurex Plus*. Remel. Part no. R30950102. Revised
11/23/07.


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5. Package Insert - Rapid Indole Reagent Droppers. Becton, Dickinson and
Company. Part no. L001237. Revision 06/2010.


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Attachment 1

Typical Growth Characteristics of strains of P. aeruginosa and is. coli (see ref. xxx).



S. aureus*

E. coli*

Gram slain reaction

(+)

(-)

Typical Growth Characteristics on Solid Media

BAP

small, circular, yellow or white,
glistening, beta hemolytic

large, round, white colonics, non-
hemolytic.

Mannitol Salt

circular, small, yellow colonics, agar
turning riuorcsccnl yellow

No Growth

XLD agar

No growth

large, flat, yellow colonics with no
black center.

L-EMB Agar

No Growth

large colonics, circular. Blue-black
with green metallic sheen

Typical Microscopic Characteristics

Cell dimensions

0.5-1.5 |im in diameter

0.5 x 1-3 |im in diameter

Cell appearance

spherical, occurring singly, in pairs
and tetrads, sometimes forming
irregular clusters

varying from almost coccoid forms to
long rods, occurring singly, in pairs
and in short chairs. Motile or non-
motile, if motile with flagella.

*After 242 hours

*After 242 hours


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SOP No. MB-27-00
Date Revised 01-16-13
Page 16 of 19

Attachment 2

Culture Initiation and Stock Culture Generation Flow Chart for S. aureus and E. coli.

 Rehydrate ampule.

i

 Transfer entire
rehydrated pellet to
TUBE A.

Ampule

Incubate

TSBor
NB

TUBE A	TUBE A

(pre-incubation) (post-incubation)

Stock Culture Generation

Inoculate TSA orNA plates with
100 ^iL culture from TUBE A;
incubate.

Harvest inoculum
from plates.

 Culture ID & Quality Control

Prepare frozen
stock cultures

BAP
/\

Selective
media

Gram Additional
Stain confirmation
steps (see
Attachment 3)

Al. Preparation of Frozen Stock Cultures. Refer to SOP MB-02 for establishment of the
organism control number.

a.	Initiate new stock cultures from lyophilized cultures of Staphylococcus aureus (ATCC
6538), and Escherichia coli (ATCC 11229) from ATCC within 18 months.

b.	Open ampule of freeze dried organism as indicated by ATCC. Using a tube containing
5-6 mL of TSB for S. aureus or NB for E. coli, aseptically withdraw 0.5 to 1.0 mL and
rehydrate the lyophilized culture. Aseptically transfer the entire rehydrated pellet back
into the original tube of broth designated as "TUBE A". Mix well.

c.	Incubate broth culture (TUBE A) at 36  1C for 24  2 hours. Record all
manipulations on the Organism Culture Tracking Form (see section 14).


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SOP No. MB-27-00
Date Revised 01-16-13
Page 17 of 19

d.	Using a sterile spreader, inoculate a sufficient number of TSA plates for S. aureus or
NA for E. coh, (e.g., 5 to 10 plates per organism) with 100 |iL each of the culture to
create a bacterial lawn. Incubate plates at 36  1C for 24  2 h.

e.	Following incubation, add 5 mL cryoprotectant solution (TSB with 15% v/v glycerol)
to the surface of each agar plate. Re-suspend the cells in this solution using a sterile
spreader or a sterile swab and aspirate the cell suspension from the surface of the agar.
Transfer the suspension into a sterile vessel.

f.	Mix the pooled contents of the vessel thoroughly. Immediately after mixing, dispense
approximately 1.0 mL aliquots into cryovials (e.g., 1.5 mL cyrovials). Perform QC of
stock cultures concurrently with freezing (see section A2: QC of Stock Cultures).

g.	Store the cryovials at -70C or below; these are the frozen stock cultures. Store stock
cultures up to 18 months. These vials are single-use only.

A2. QC of Stock Cultures.

a.	Conduct QC of the pooled culture concurrently with freezing. Streak a loopful on a
plate of BAP. In addition, for S. aureus streak a loopful onto selective media (i.e.,
MSA); for E. coli, streak a loopful onto XLD. Incubate all plates at 36  1C for 24  2
hours.

b.	Following the incubation period, record the colony morphology as observed on the
BAPs and selective media plates (including the absence of growth) and Gram stain.
See Attachment 1 for details on cell and colony morphology, colony characteristics on
selective media, and stain reactions.

c.	For each organism, perform a Gram stain from growth taken from the BAPs according
to the manufacturer's instructions. Observe the Gram reaction by using brightfield
microscopy at 1000X magnification (oil immersion).

d.	For additional confirmation steps refer to the appropriate Confirmation Flow Chart for
S. aureus and E. coli (see Attachment 3). Refer to 15.3-15.5 for instructions.

e.	Record all confirmation results on the Test Microbe Confirmation Sheet (Quality
Control) (see section 14).


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SOP No. MB-27-00
Date Revised 01-16-13
Page 18 of 19

Attachment 3

Confirmation Flow Chart for S. aureus

S. aureus Identification

Identified as S1. aureus


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SOP No. MB-27-00
Date Revised 01-16-13
Page 19 of 19

Attachment 3 (cont.)

Confirmation Flow Chart for is. coli

E. coli Identification

Take one colony from sample
plate onto BAP and XLD.
Confirm GNR

1

BAP - Round colonies, non -hemolytic
XLD - Yellow colonies. No black center.

Perform Spot Indole test from BAP colony
(Positive result = pink coloration in area
with culture)

Not E. coli

Negative

Positive

E. coli


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